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. 2011 Apr 18;6(4):e18773.
doi: 10.1371/journal.pone.0018773.

Latency antigen α-crystallin based vaccination imparts a robust protection against TB by modulating the dynamics of pulmonary cytokines

Bappaditya Dey  1 , Ruchi Jain, Aparna Khera, Umesh D Gupta, V M Katoch, V D Ramanathan, Anil K Tyagi
Affiliations

Latency antigen α-crystallin based vaccination imparts a robust protection against TB by modulating the dynamics of pulmonary cytokines

Bappaditya Dey et al. PLoS One. .

Abstract

Background: Efficient control of tuberculosis (TB) requires development of strategies that can enhance efficacy of the existing vaccine Mycobacterium bovis Bacille Calmette Guerin (BCG). To date only a few studies have explored the potential of latency-associated antigens to augment the immunogenicity of BCG.

Methods/principal findings: We evaluated the protective efficacy of a heterologous prime boost approach based on recombinant BCG and DNA vaccines targeting α-crystallin, a prominent latency antigen. We show that "rBCG prime-DNA boost" strategy (R/D) confers a markedly superior protection along with reduced pathology in comparison to BCG vaccination in guinea pigs (565 fold and 45 fold reduced CFU in lungs and spleen, respectively, in comparison to BCG vaccination). In addition, R/D regimen also confers enhanced protection in mice. Our results in guinea pig model show a distinct association of enhanced protection with an increased level of interleukin (IL)12 and a simultaneous increase in immuno-regulatory cytokines such as transforming growth factor (TGF)β and IL10 in lungs. The T cell effector functions, which could not be measured in guinea pigs due to technical limitations, were characterized in mice by multi-parameter flow cytometry. We show that R/D regimen elicits a heightened multi-functional CD4 Th1 cell response leading to enhanced protection.

Conclusions/significance: These results clearly indicate the superiority of α-crystallin based R/D regimen over BCG. Our observations from guinea pig studies indicate a crucial role of IL12, IL10 and TGFβ in vaccine-induced protection. Further, characterization of T cell responses in mice demonstrates that protection against TB is predictable by the frequency of CD4 T cells simultaneously producing interferon (IFN)γ, tumor necrosis factor (TNF)α and IL2. We anticipate that this study will not only contribute toward the development of a superior alternative to BCG, but will also stimulate designing of TB vaccines based on latency antigens.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Development of rBCG strain over-expressing α-crystallin and analysis of expression and in vitro growth.
(A) A Mycobacteria - Escherichia coli shuttle plasmid pSD5.hsp65.acr was engineered to over-express α-crystallin under transcriptional control of the promoter of hsp65 gene of M. leprae. The recombinant plasmid was electroporated into M. bovis BCG to generate rBCGacr strain. (B) The whole cell lysates of BCG and rBCGacr (grown to A600 nm of 0.8–1.0) were analysed for the expression of α-crystallin by SDS-PAGE and immuno-blotting by using antigen specific antibodies. By densitometric analysis, rBCGacr was found to produce a considerably higher level of 16 kDa α-crystallin protein in comparison to wild type BCG strain (∼15 fold). (C) Growth kinetics of BCG and rBCG during 19 days of culture in 7H9 medium. The A600 nm of broth culture was plotted against time. Data points are presented as mean ± SE of duplicate cultures.
Figure 2
Figure 2. Protection by α-crystallin based prime boost regimens against M. tuberculosis challenge.
The figure depicts the bacillary load in lungs and spleen of guinea pigs at (A) 10 weeks (n = 5) and (B) 16 weeks (n = 6) post-infection. Animals vaccinated with R/D regimen exhibited a significantly lower bacillary load in lung and spleen when compared to the unvaccinated as well as BCG vaccinated animals at both 10 weeks and 16 weeks post-infection. Log10 CFU is represented by box plot, wherein median values are denoted by horizontal line, the mean is represented by ‘+’, inter quartile range by boxes, and the maximum and minimum values by whiskers. R/D, rBCG prime – DNAacr boost; R/V, rBCG prime – vector boost. (*, p<0.05, **, p<0.01 and ***, p<0.001, when compared to the saline group, One-way ANOVA).
Figure 3
Figure 3. α-crystallin based prime boost vaccination reduces antigen load in pulmonary granulomas.
The representative photomicrographs of lung sections show immuno-histochemical staining (brown color) for Ag85 complex proteins in pulmonary granulomas at (A) 10 weeks and (B) 16 weeks post-infection. (A) Animals in the unvaccinated group exhibited extensive staining within the granulomatous regions; BCG and rBCGacr vaccinated animals showed moderate and comparable staining; animals vaccinated with R/D regimen showed a reduced antigen staining, when compared to BCG vaccinated animals. (B) Unvaccinated animals and BCG vaccinated animals showed similar staining pattern as observed at 10 weeks; rBCGacr and R/D vaccinated animals showed a significantly reduced antigen load. Scale bar represents 1 mm. Extent (Q) of staining was measured by light microscopy [Q = intensity (I)×area (A) of staining] and represented graphically as median (± inter quartile range). R/D, rBCG prime – DNAacr boost. (*, p<0.05; **, p<0.01, when compared to the unvaccinated animals, Mann-Whitney U test).
Figure 4
Figure 4. Reduction in gross pathological damage by α-crystallin based prime boost regimen.
The figure depicts representative photographs and graphical depiction of gross scores of lung, liver and spleen of guinea pigs at (A) 10 weeks (n = 5) and (B) 16 weeks (n = 6) post-infection. Based on the extent of involvement, number and size of tubercles, areas of inflammation and necrosis; gross pathological scores were graded from 1–4 as described in materials and methods and represented graphically. (A) BCG, rBCGacr and R/D regimens resulted in a significant reduction in pathological lesions in lung and liver compared to the saline control; animals in R/D group also showed a significantly reduced spleen pathology, when compared to BCG vaccinated animals. (B) Animals in R/D group showed minimal pathology in all the organs, when compared to BCG vaccinated animals. Each point represents score for an individual animal and the bar depicts median (± inter quartile range) for each group. Missing data points represent the animals that succumbed to disease before euthanasia. R/D, rBCG prime – DNAacr boost; R/V, rBCG prime – vector boost. (*, p<0.05 and **, p<0.01, when compared to the saline group, Mann-Whitney U test).
Figure 5
Figure 5. Reduced granulomatous inflammation following M. tuberculosis infection in animals vaccinated with R/D regimen.
Representative photomicrographs of H&E stained lung and liver sections showing granulomatous pathology at (A) 10 weeks (n = 5) and (B) 16 weeks (n = 6) post-infection. Lung: at both the time points, unvaccinated animals showed the presence of coalescing granulomas with extensive necrosis; BCG and rBCG groups showed moderate involvement with well-organized discrete granulomas with or without central necrosis; animals in R/D group showed reduced granulomatous infiltration with a few diffused aggregates of inflammatory cells in the peribronchial and perivascular areas. Liver: unvaccinated animals showed moderate to high granulomatous lesions; BCG vaccinated animals showed minimal inflammatory aggregates; all the regimens based on α-crystallin showed negligible hepatic inflammation. Scale bar represents 2 mm. Granuloma % were measured by light microscopy and graphically represented by box plot (notations are described in the legend of Fig. 1). R/D, rBCG prime – DNAacr boost; R/V, rBCG prime – vector boost. (*, p<0.05, **, p<0.01, when compared to the saline group, Mann-Whitney U test).
Figure 6
Figure 6. Influence of α-crystallin based prime boost vaccination on pulmonary fibrosis following M. tuberculosis infection.
The figure depicts representative photomicrographs of Van Gieson stained lung sections of guinea pigs euthanized at (A) 10 weeks and (B) 16 weeks post-infection. (A) Unvaccinated animals showed extensive fibrosis characterized by widespread presence of thick bands of collagen fibers (red color) in the granulomatous areas; BCG and rBCGacr vaccinated animals showed a moderate staining with the presence of thin bands of collagen. Animals vaccinated with R/D regimen showed no evident signs of collagen staining other than the usual occurrence of collagen in the peri-bronchial and peri-vascular areas. (B) Unvaccinated animals and BCG vaccinated animals showed similar staining pattern as observed at 10 weeks; rBCGacr and R/D vaccinated animals showed negligible staining. Scale bar represents 1 mm. Extent (Q) of pulmonary fibrosis was measured by light microscopy [Q = Intensity (I)×area (A) of staining] and represented graphically as median (± inter quartile range). R/D, rBCG prime – DNAacr boost. (*, p<0.05; **, p<0.01, when compared to the unvaccinated animals, Mann-Whitney U test).
Figure 7
Figure 7. α-crystallin based prime boost vaccination induces dynamic changes in the cytokine milieu in lungs.
Expression of various cytokines was measured in the lung tissues of vaccinated and saline treated guinea pigs at (A) 10 weeks and (B) 16 weeks post-infection by semi-quantitative real time RT-PCR by using gene specific primers. The data were normalized to 18S rRNA levels and then normalized to the values of uninfected animals to obtain ΔΔCt values. The % fold induction values were measured [2−ΔΔCt×100] and were graphically represented as mean (± SE). For cytokine measurement, 3 lung samples were chosen randomly from each group. R/D, rBCG prime – DNAacr boost. (*, p<0.05 and **, p<0.01, when compared to the saline group, One-way ANOVA).
Figure 8
Figure 8. Induction of CD4 Th1 cell responses by R/D immunization.
At 12 weeks post-immunization, T lymphocytes were purified from PPD and α-crystallin stimulated splenocytes (pooled from four mice per group) and stained for cell surface marker (CD4) along with intracellular staining for IFNγ, TNFα and IL2 followed by FACS analysis. Frequency of IFNγ, TNFα and IL2 producing cells was determined on CD4 T cell gated population. (A) Total frequency (%) of PPD and α-crystallin specific cytokine producing CD4 T cells in spleen. (B) Frequency (%) of PPD and α-crystallin specific CD4 T cells expressing each of the seven combinations of IFNγ, TNFα and IL2. (C) Proportion of PPD and α-crystallin specific CD4 T cells producing one, two or three cytokines. (D) Frequency (%) of PPD and α-crystallin specific 3+ CD4 T cells in spleen along with MFI and iMFI for IFNγ, TNFα and IL2. R/D, rBCG prime – DNAacr boost.
Figure 9
Figure 9. α-crystallin based vaccination provides protection against M. tuberculosis challenge in mice.
Mice were infected 12 weeks after primary immunization and euthanized at 2, 4 and 10 weeks post-infection and lung and spleen bacillary load were determined. Log10 CFU is graphically represented by box plot (notations are described in the legend of Fig. 1). Animals vaccinated with R/D regimen exhibited a significantly lower bacillary load in lung and spleen when compared to the unvaccinated as well as BCG vaccinated animals at 2, 4 and 10 weeks post-infection. R/D, rBCG prime – DNAacr boost. (*, p<0.05, **, p<0.01 and ***, p<0.001, when compared to the saline group, One-way ANOVA).
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